No abstract

In interphase Chinese hamster ovary (CHO) cells, the centrosome is attached to the nucleus very firmly . This nuclear-centrosome complex is isolated as a coherent structure by lysis and extraction of cells with Triton X-100 in a low ionic strength medium . Under these conditions, the ultrastructure of the centrioles attached to the nucleus can be discerned by electron microscopy of whole-mount preparations . The structural changes of the centrioles as a function of the cell cycle were monitored by this technique. Specifically, centriolar profiles were placed into six categories according to their orientation and the length ratio of daughter and parent centrioles . The proportion of centrioles in each category was plotted as a frequency histogram. The morphological changes in the centriole cycle were characterized by three distinguishable events : nucleation, elongation, and disorientation . The progress of centrioles through these stages was determined in synchronous populations of cells starting from S or M phase, in cells inhibited in DNA synthesis by addition of thymidine, and in cytoplasts . The results provide a quantitative description of the events of the centriole cycle. They also show that, in complete cells, nucleation, elongation, and disorientation are not dependent upon DNA synthesis. However, in cytoplasts, although elongation and disorientation occur as in normal cells, nucleation is blocked . Procentriole formation appeared to be inhibited by the removal of the nucleus . We suggest that coordination of centriole replication and nuclear replication may depend upon a signal arising from the nucleus.The centrosome consists of a pair of centrioles and pericentriolar material and serves as a mitotic center in animal cells (21) .The reproduction of the mitotic centers is coordinated with other events in the cell cycle and has been dissected functionally into distinct phases of duplication and splitting or separation (12).With the use of synchronized tissue culture cells and electron microscopy of thin sections, the centriole cycle has been described qualitatively in structural terms (17,19) . Daughter cells formed by a cell division each receive a pair of orthogonally oriented centrioles. The two centrioles become disoriented and lose their orthogonal arrangement in G L phase . During late G L or S phase, a short daughter centriole, the procentriole, appears at the proximal end oriented orthogonally with respect to the parent . Formation of the procentriole may be considered a nucleation event . The procentriole elongates slowly through S and G2 phase and attains almost full size at prophase when the two centriolar pairs separate and begin to migrate towards opposite ends of the nucleus . The daughter centriole generally does not attain its full length until early G L of the next cell cycle . A pair of orthogonally oriented centrioles is positioned at each spindle pole and is segregated to each daughter cell by mitosis, after which a new centriole cycle is begun . Although different cell types...

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Ceils were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate.

Abstract. During early development, intracellularCa 2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca 2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca 2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca 2+ removal, but were blocked by intracellular Ca 2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca 2+ and showed no associated Ca ~+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca 2+ transient and was dependent on Ca 2 § mobilization. We conclude that GVBD is Ca 2+ independent, but that the dependence of NEBD on Ca 2+ suggests regulation by more than one pathway. As cells develop from Ca2+-independent germinal vesicle oocytes to internal Ca 2 § pronuclear eggs, internal Ca 2 § pools increase by approximately fourfold.

Using the transgenic AEQUORlN system, we showed that the cotyledons and leaves of Arabidopsis thaliana seedlings developed a biphasic luminescence response to anoxia, indicating changes in cytosolic Ca2+ levels. A fast and transient luminescence peak occurred within minutes of anoxia, followed by a second, prolonged luminescence response that lasted 1.5 to 4 h. l h e Ca2+ channel blockers Cd3+, La3+, and ruthenium red (RR) partially inhibited the first response and promoted a larger and earlier second response, suggesting different origins for these responses. Both Cd3+ and RR also partially inhibited anaerobic induction of alcohol dehydrogenase gene expression. However, although anaerobic alcohol dehydrogenase gene induction occurred in seedlings exposed to wateragar medium and in roots, related luminescence responses were absent. Upon return to normoxia, the luminescence of cotyledons, leaves, and roots dropped quickly, before increasing again in a Gd3+-, La3+-, ethyleneglycol-bis(P-aminoethyl ether)-N,N'-tetraacetic acid-, and RR-sensitive fashion.When exposed to anoxia, plants undergo major metabolic changes to maintain energy production despite a shut off of respiratory phosphorylation. They do so by increasing the rate of Suc and starch mobilization, by accelerating the rate of glycolysis and diversifying its end products, and by accelerating the ethanol fermentation pathway (reviewed by Ricard et al., 1994). These metabolic changes are associated with major changes in gene expression involving a decrease in general mRNA translatability and an activation of expression of a set of anoxic genes, most of which code for enzymes involved in starch and Glc mobilization, glycolysis, and ethanol fermentation (Sachs et al